Summary The incidence of acute myeloid leukemia (AML) increases through age, and as the population of the developed world ages, the overall societal incidence will also rise. AML is a genetically heterogeneous disease with molecular abnormalities being observed that influence a wide array of cellular processes. There is substantial need to better understand the processes governing AML therapeutic resistance and to develop better therapeutic strategies. Sphingolipids have gained interest for their roles in cell death and proliferation. Ceramide is a bioactive sphingolipid that can promote cell death. Not surprisingly, various anticancer therapies have been shown to stimulate ceramide accumulation and upregulation of ceramide metabolism has been associated with therapeutic resistance. We and others have worked to develop the ceramide nanoliposome (Lip-C6) as an anticancer therapy that delivers the C6-ceramide analog to malignant cells. Lip-C6 is presently in a phase I clinical trial for solid tumor malignancies (ClinicalTrials.gov identifier: NCT02834611). Unfortunately, resistance to Lip-C6 can develop due to enhanced ceramide metabolism. Long-term, the goal of this proposal is to improve the anti-AML efficacy of therapeutics such as Lip-C6 by identifying and targeting pathways, such as dysfunctional epigenetics, that limit their effectiveness. Therefore, the overarching objective of this proposal is to identify epigenetic-linked sphingolipid metabolic dysfunction as a mediator of resistance to Lip-C6 therapy in AML. Our preliminary studies show that De Novo AML (DN-AML), such as FLT3ITD-mutated AML, is resistant to Lip-C6 therapy and can be defined by an increase in overall sphingolipid metabolic flux. Secondly, this application will study the anti-AML efficacy of the hypomethylating agent decitabine in combination with Lip-C6. The initial aim of this proposal is to link dysfunctional sphingolipid metabolism in AML with aberrant DNA methylation. This will test the hypothesis that resistance to Lip-C6 in of AML can be due to epigenetic dysfunction that upregulates pathways of ceramide metabolism. This is significant given that resistance to ceramide-elevation by Lip-C6 is prominent in DN-AML where alterations to epigenetic regulators can occur. Importantly, the pro-apoptotic sphingolipid ceramide can also be elevated by standard care chemotherapeutics, which highlights the relevance and importance of this metabolic pathway of resistance. The second aim of this proposal is to study the combinatorial anti-AML efficacy of decitabine and Lip-C6. This aim hypothesizes that DNA hypomethylating agents, such as decitabine, can restore the anti-AML efficacy of Lip-C6 for AML with epigenetic dysfunction. Overall, this is important because hypomethylating agents may overcome therapeutic resistance that persists in DN-AML due to epigenetic-driven ceramide metabolism. Overall, this research will advance our understanding of a cooperative therapeutic resistance mechanism involving sphingolipids and epigenetic dysfunction in AML, which will serve as the basis to develop better combinatorial therapeutic approaches.